Site-specific incorporation of unnatural amino acids into proteins by cell-free protein synthesis

Abstract

Cell-free protein synthesis (CFPS) offers a fast and inexpensive means to incorporate unnatural amino acids (UAAs) site specifically into proteins. This enables engineering of proteins and allows production of protein-based probes for analysis of their interactions with other molecules. Using dialysis Escherichia coli CFPS system in combination with aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii high expression yield of proteins with site specifically incorporated UAAs can be achieved. Typically the target protein can be prepared at concentrations of about 1 mg/mL, which is generally sufficient for subsequent applications. © 2014 Springer Science+Business Media, LLC.